Double mutant alpha-7 nicotinic acetylcholine receptor

ABSTRACT

The invention relates to a novel methods for measuring cellular ion channel transmission and methods and compositions useful in the identification of ligand gated ion channel agonists and modulators.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the following provisionalapplication: U.S. Ser. No. 60/136,174 filed May 27, 1999 under 35 U.S.C119(e)(1).

FIELD OF THE INVENTION

The invention relates to a novel methods for measuring cellular ionchannel transmission and methods and compositions useful in theidentification of ligand gated ion channel agonists and modulators.

BACKGROUND OF THE INVENTION Ion Channels

Ion channel proteins form hydrophilic pores that extend across thecellular lipid bilayer; when these pores are open, they allow specificmolecules (usually inorganic ions of appropriate size and charge) topass through them and thereby cross the membrane.

Channel proteins which are concerned specifically with inorganic iontransport are referred to as ion channels, and include ion channels forsodium, potassium, calcium, and chloride ions. Ion channels which openin response to a change in the voltage across the membrane are referredto as voltage gated ion channels (or voltage-dependent ion channels).Ion channels which open in response to the binding of a ligand to thechannel protein are referred to as ligand gated ion channels.

The present invention describes new ion channels and provides methodsand compositions suitable for high throughput screening of ion channels.

DESCRIPTION OF THE INVENTION Voltage Gated Ion Channels

Voltage Gated Sodium Channel

Voltage gated ion channels are a class of channel proteins that play amajor role in cellular electrical excitability. In the majority ofexcitable tissues, the early depolarization phase of action potentialsis mediated by a sodium current via voltage-dependent sodium channels(also known as voltage-gated sodium channels or VGSCs). The sodiumchannel is one of the most thoroughly characterized of the voltage gatedchannels. The primary structures of many sodium channels from a varietyof tissues (brain, skeletal muscle and cardiac muscle) and organisms(jellyfish, squid, eel, rat, human) have been identified, and theiramino acid sequences show individual regions which are highly conservedover evolution, indicating that voltage-dependent sodium channels belongto a large superfamily of evolutionarily related proteins. All publishedpolypeptide complexes of VGSCs have in common a large, about 260 kDaglycoprotein (the pore forming subunit) which is called the alphasubunit (Agnew et al. 1978; Agnew et al. 1980; Catterall 1986; Catterall1992). Additional lower molecular weight polypeptides, thebeta-subunits, have been found to be associated with sodium channelsfrom mammalian muscle (Kraner et al. 1985; Tanaka et al. 1983) and brain(Hartshorne and Catterall 1984). The large, pore-forming alpha subunitis sufficient for all known functions of VGSCs (Catterall 1992) whilethe beta subunits modulate some of the functions of the alpha subunit(Catterall 1992).

Voltage Gated Potassium Channels

Voltage-gated potassium channels make up a large molecular family ofintegral membrane proteins that are fundamentally involved in thegeneration of bioelectric signals such as nerve impulses. These proteinsspan the cell membrane, forming potassium-selective pores that arerapidly switched open or closed by changes in membrane voltage. Severalchemical entities have been discovered to be potent and specific openersof vascular potassium K+ channels. These include cromakalim and itsderivatives and RP 52891. This mechanism is also shared, at leastpartially, by drugs such as minoxidil, diazoxide, pinacidil andnicorandil. The opening of plasmalemmal K+ channels produces loss ofcytosolic K+. This effect results in cellular hyperpolarization andfunctional vasorelaxation. In normotensive or hypertensive rats, K+channel activators decrease aortic blood pressure (by producing adirectly mediated fall in systemic vascular resistance) and reflexivelyincrease heart rate. K+ channel openers produce selective coronaryvasodilatation and afford functional and biochemical protection to theischemic myocardium.

The structure of a typical voltage-gated potassium channel protein isknown to be comprised of six membrane spanning domains in each subunit,each of which is regulated by changes in membrane potential. B. Hille.“Ionic Channels of Excitable Membranes”(Sinauer, Sunderland, Mass.,1992). Voltage-gated potassium channels sense changes in membranepotential and move potassium ions in response to this alteration in thecell membrane potential. Molecular cloning studies on potassium channelproteins has yielded information primarily for members of thevoltage-gated family of potassium channels. Various genes encoding thesevoltage-gated family of potassium channel proteins have been clonedusing Drosophila genes derived from both the Shaker, Shaw and Shab loci;Wei, A. et. al., Science (1990) Vol. 248 pp. 599-603.

Voltage Gated Calcium Channels

Voltage-gated calcium channels are present in neurons, and in cardiac,smooth, and skeletal muscle and other excitable cells. These channelsare known to be involved in membrane excitability, muscle contraction,and cellular secretion, such as in exocytotic synaptic transmission(McCleskey, et al.,1987). In neuronal cells, voltage-gated calciumchannels have been classified by their electrophysiological as well asby their biochemical (binding) properties.

Calcium channels are generally classified according to theirelectrophysiological properties as Low-voltage-activated (LVA) orHigh-voltage-activated (HVA) channels. HVA channels are currently knownto comprise at least three groups of channels, known as L-, N- andP-type channels (Nowycky, et al., 1985). These channels have beendistinguished one from another structurally and electrophysiologicallyas well as biochemically on the basis of their pharmacology and ligandbinding properties. Thus, dihydropyridines, diphenylalkylamines andpiperidines bind to the alpha1 subunit of the L-type calcium channel andblock a proportion of HVA calcium currents in neuronal tissue, which aretermed L-type calcium currents.

N- or omega-type HVA calcium channels are distinguishable from othercalcium channels by their sensitivity to omega conotoxins (omegaconopeptides). Such channels are insensitive to dihydropyridinecompounds, such as L-type calcium channel blockers nimodipine andnifedipine. (Sher and Clementi, 1991).

Ligand Gated Ion Channel Receptors

Ligand-gated ion channels provide a means for communication betweencells of the central nervous system. These channels convert a signal(e.g., a chemical referred to as a neurotransmitter) that is released byone cell into an electrical signal that propagates along a target cellmembrane. A variety of neurotransmitters and neurotransmitter receptorsexist in the central and peripheral nervous systems. At the presenttime, numerous families of ligand-gated receptors have been identifiedand characterized on the basis of sequence identity these includenicotinic acetylcholine, glutamate, glycine, GABA A, 5-HT3, and thepurinoceptors. These can be further characterized by whether the gatedion channel transmits cations or anions. Those which form cationicchannels include, for example, excitatory nicotinic acetylcholinereceptors (nAChRs), excitatory glutamate-activated receptors, the 5-HT3serotonin receptor, and the purine receptor.

Those which form anionic channels include, for example, the inhibitoryGABA and glycine-activated receptors. This discussion will confineitself to those ligand gated ion channel receptors which conductcations.

5HT₃ Receptor

Molecular cloning has indicated that serotonin (5-hydroxytryptamine,also referred to as 5-HT) receptors belong to at least two proteinsuperfamilies: G-protein-associated receptors and ligand-gated ionchannel. The 5-HT₃ receptor belongs to the family of ligand-gated ionchannels. As discussed below the 5-HT₃ receptor is primarily a sodiumpotassium ligand gated ion channel under physiologic conditions. Theinflammatory and painproducing effects of serotonin are generallybelieved to be mediated via 5HT₃ receptors on peripheral sensory endings(Richardson, B. P., et al., 1985).

Nicotinic Receptors

The nicotinic acetylcholine receptors (nAChRs) are multisubunit proteinsof neuromuscular and neuronal origins. These receptors form ligand-gatedion channels that mediate synaptic transmission between nerve and muscleand between neurons upon interaction with the neurotransmitteracetylcholine (ACh). Since various nicotinic acetylcholine receptor(nAChR) subunits exist, a variety of nAChR compositions (i.e.,combinations of subunits) exist. The different nAChR compositionsexhibit different specificities for various ligands and are therebypharmacologically distinguishable. Thus, the nicotinic acetylcholinereceptors expressed at the vertebrate neuromuscular junction invertebrate sympathetic ganglia and in the vertebrate central nervoussystem have been distinguished on the basis of the effects of variousligands that bind to different nAChR compositions. For example, theelapid alpha-neurotoxins that block activation of nicotinicacetylcholine receptors at the neuromuscular junction do not blockactivation of some neuronal nicotinic acetylcholine receptors that areexpressed on several different neuron-derived cell lines.

Muscle nAChR is a glycoprotein composed of five subunits with thestoichiometry alpha 2 alpha (gamma or epsilon) delta. Each of thesubunits has a mass of about 50-60 kilodaltons (kd) and is encoded by adifferent gene. The alpha 2 beta (gamma or epsilon) delta complex formsfunctional receptors containing two ligand binding sites and aligand-gated transmembrane channel. Upon interaction with a cholinergicagonist, muscle nicotinic AChRs conduct sodium ions. The influx ofsodium ions rapidly short-circuits the normal ionic gradient maintainedacross the plasma membrane, thereby depolarizing the membrane. Byreducing the potential difference across the membrane, a chemical signalis transduced into an electrical signal that signals muscle contractionat the neuromuscular junction.

Functional muscle nicotinic acetylcholine receptors have been formedwith alpha beta delta gamma subunits, alpha beta gamma subunits, alphabeta delta subunits, alpha beta gamma subunits or alpha delta subunits,but not with only one subunit (see e.g., Kurosaki et al. 1987; Camachoet al. 1993) In contrast, functional neuronal AChRs (nAChRs) can beformed from alpha subunits alone or combinations of alpha and betasubunits. The larger alpha subunit is generally believed to be theACh-binding subunit and the lower molecular weight beta subunit isgenerally believed to be the structural subunit, although it has notbeen definitively demonstrated that the beta subunit does not have theability to bind ACh. Each of the subunits which participate in theformation of a functional ion channel are, to the extent they contributeto the structure of the resulting channel, “structural” subunits,regardless of their ability (or inability) to bind ACh.

Neuronal AChRs (nAChRs), which are also ligand-gated ion channels, areexpressed in ganglia of the autonomic nervous system and in the centralnervous system (where they mediate signal transmission), inpost-synaptic locations (where they modulate transmission), and in pre-and extra-synaptic locations (where they may have additional functions).The nAChRs comprise a large family of neurotransmitter regulated ionchannels that control neuronal activity and brain function. Thesereceptors have a pentameric structure. The gene family is composed ofnine alpha and four beta subunits that co-assemble to form multiplesubtypes of receptors that have a distinctive pharmacology. Acetycholineis the endogenous regulator of all of the subtypes, while nicotinenon-selectively activates all nAChRs. Known chemical templates havesubtype selectivity.

α7 nAChR is a ligand-gated Ca⁺⁺ channel formed by a homopentamer of α7subunits. α7 nAChR is of particular interest because α7 nAChR agonistsincrease neurotransmitter release, increase cognition, arousal,attention, learning and memory. α7 nAChR is expressed at high levels inthe hippocampus, ventral tegmental area and ascending cholinergicprojections from nucleus basilis to thalamocortical areas. Previousstudies have established that a α-bungarotoxin (α-btx) binds selectivelyto this homopetameric, α7 nAChR subtype, and that α7 nAChR has a highaffinity binding site for both α-btx and methyllycaconitine (MLA). Wehave chosen to use α7 nAChR as a model system for high throughput drugscreening

Glutamate Receptors

Glycine also functions in excitatory transmission by modulating theactions of glutamate, the major excitatory neurotransmitter in thecentral nervous system. (Johnson and Ascher, 1987)

Glutamate binds or interacts with one or more glutamate receptors whichcan be differentiated pharmacologically into several subtypes. In themammalian central nervous system (CNS) there are three main subtypes ofionotropic glutamate receptors, defined pharmacologically by theselective agonists N-methyl-D-aspartate (NMDA), kainate (KA), andalpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). TheNMDA receptor has been implicated in a variety of neurologicalpathologies including stroke, head trauma, spinal cord injury, epilepsy,anxiety, and neurodegenerative diseases such as Alzheimer's Disease(Watkins and Collingridge 1989). A role for NMDA receptors innociception and analgesia has been postulated as well (Dickenson, 1990).More recently, AMPA receptors have been widely studied for theirpossible contributions to such neurological pathologies (Fisher andBogousslavsky, 1993).

When activated by glutamate, the endogenous neurotransmitter, the NMDAreceptor permits the influx of extracellular calcium (Ca++) and sodium(Na+) through an associated ion channel. The NMDA receptor allowsconsiderably more influx of Ca++ than do kainate or AMPA receptors andis an example of a receptor-operated Ca++ channel. Normally, the channelis opened only briefly, allowing a localized and transient increase inthe concentration of intracellular Calcium (Ca++) which, in turn, altersthe functional activity of the cell.

The activity of the NMDA receptor-ionophore complex is regulated by avariety of modulatory sites that can be targeted by selectiveantagonists. Competitive antagonists, such as the phosphonate AP5, actat the glutamate binding site, whereas noncompetitive antagonists, suchas phencyclidine (PCP), MK-801 or magnesium (Mg++), act within theassociated ion channel (ionophore). There is also a glycine binding sitethat can be blocked selectively with compounds such as 7-chlorokynurenicacid. There is evidence suggesting that glycine acts as a co-agonist, sothat both glutamate and glycine are necessary to fully elicit NMDAreceptor-mediated responses. Other potential sites for modulation ofNMDA receptor function include a zinc (Zn<2+>) binding site and a sigmaligand binding site. Additionally, endogenous polyamines such asspermine are believed to bind to a specific site and so potentiate NMDAreceptor function (Ransom and Stec, 1988). The potentiating effect ofpolyamines on NMDA receptor function may be mediated via a specificreceptor site for polyamines.

Purinergic Receptors

Purinergic receptors are classified as P1 (adenosine as ligand) and P2(ATP as ligand). The P2 receptors are subclassified into two broadtypes-those that are 7-transmembrane receptors that couple to G-proteins(P 2Y, P 2U, P 2T, and perhaps P 2Z. Another major class ofpurinoceptors are the P2x purinoceptors which are ligand-gated ionchannels possessing intrinsic ion channels permeable to Na+, K+, andCa++. P2x receptors described in sensory neurons are important forprimary afferent neurotransmission and nociception. ATP is known todepolarize sensory neurons and plays a role in nociceptor activationsince ATP released from damaged cells stimulates P2x receptors leadingto depolarization of nociceptive nerve-fiber terminals. ATP-sensitivepotassium channels have been discovered in numerous tissues, includingkidney, vascular and non-vascular smooth muscle and brain, and bindingstudies using radiolabeled ligands have confirmed their existence.Opening of these channels causes potassium (K<+>) efflux andhyperpolarizes the cell membrane

Ion Channels as Drug Targets

Ion channels both ligand gated and voltage gated, are in generalexcellent and validated drug targets. For some channels however, afunctional high throughput screening assay is problematic becauseexpression levels are low and function is hard to measure using standarddetection technology for high throughput screening. For those channelswhich normally conduct a cation other than calcium high througputscreening methods are often cumbersome. For calcium conductance however,several rapid assays exist. It would often be desireable to Thisinvention provides the scientist with a detailed description about howto convert a channel normally conducting sodium or potassium underphysiologic conditions to one conducting calcium for ease in assaydevelopment.

The α7 nAChR discussed above is one ligand gated ion channel that hasproved to be a difficult target for developing a functional highthroughput screening assay. Native α7 nAChR are not routinely able to bestably expressed in most mammalian cell lines (Cooper and Millar 1997).Repeated attempts by our group to stably express the human α7 nAChR inHRK 293, CHO, COS and SH-EP1 were unsuccessful. While it was possible toidentify cell lines that initially expressed functional α7 nAChR, theselines dramatically lost receptor expression with prolonged growth inculture. Under these conditions it was not possible to use these linesfor screening purposes. Another feature that makes functional assays ofα7 nAChR challenging is that the receptor is rapidly (100 milliseconds)inactivated agonist application. This rapid inactivation greatly limitsthe functional assays that can be used to measure channel activity

One solution to the problem is to engineer the α7 nAChR to have a longerduration of open probability and to have it be expressed better inmammalian cells. We are aware of a report indicating that a chimericreceptor formed between the N-terminal ligand binding domain of the α7nAChR (AA 1-201) and the pore forming C-terminal domain of the 5-HT₃receptor expressed well in Xenopus oocytes while retaining nicotinicagonist sensitivity (Eisele et al. 1993). Eisele et al (1993) used theN-terminus of the avian (chick) form of the α7 nAChR receptor and thec-terminus of the mouse form of the 5-HT₃ gene. The report of Eisele et.al. was interesting to us because we knew from our own studies that the5-HT₃ channels expressed well in most mammalian cells. In addition, wealso knew from past studies that 5-HT₃ channels inactivated much slowerthan nicotinic channels. A chimeric receptor prepared from the ligandbinding region of α7 nAChR and the pore forming domain of 5-HT₃ mightexpress well in mammalian cells and might be easier to measure in afunctional assay. However, under physiological conditions the α7 nAChRis a calcium channel while the 5-HT₃ receptor is a sodium and potassiumchannel. Indeed, Eisele et al. teaches that the chicken α7 nAChR/ mouse5-HT₃ receptor behaves quite differently than the native α7 nAChR withthe pore element not conducting calcium but actually being blocked bycalcium ions. The chicken/mouse hybrid of Eisele is also not suitablefor accessing compounds for their activity at the human α7 nAChRreceptor. The human α7 nAChR has 92% identity with the chicken α7 nAChR,but surprisingly, the pharmacology of the two receptors are different.For example, 1,1-dimethyl-4-phenylpiperazinium is a full agonist at thehuman receptor and a partial agonist at the chicken receptor (Peng et al1994). Other large species-specific differences in binding affinity havebeen noted (Peng et al 1994).

Ligand binding can be accessed in either whole cells or membranepreparations but both kinds of assays are cumbersome. Whole cell assayshave been difficult to perform in a high throughput screening formatbecause of the extensive washing and manipulation required to obtain agood signal to noise ratio. Isolated membranes have been used in suchassays but also typically require extensive manipulation to prepare themembranes themselves and the assay itself requires extensivemanipulation and washing to obtain a favorable signal to noise ratio.Such assays are illustrated in U.S. Pat. No. 6,022,704. A binding assaywhich could be performed without such required extensive manipulationwould be extremely useful.

Within the last few years very precise measurement of cellularfluorescence in a high throughput whole cell assay has become possiblewith the use of a device marketed by Molecular Devices, Inc. designated“FLIPR” (Schroeder et al. 1996), entire document, full referenceprovided below, incorporated herein by reference. FLIPR has shownconsiderable utility in measuring membrane potential of mammalian cellsusing voltage-sensitive fluorescent dyes but is useful for measuringessentially any cellular fluorescence phenomenon. The device uses lowangle laser scanning illumination and a mask to selectively excitefluorescence within approximately 200 microns of the bottoms of thewells in standard 96 well plates. The low angle of the laser reducesbackground by selectively directing the light to the cell monolayer.This avoids background fluorescence of the surrounding media. Thissystem then uses a CCD camera to image the whole area of the platebottom to measure the resulting fluorescence at the bottom of each well.The signal measured is averaged over the area of the well and thusmeasures the average response of a population of cells. The system hasthe advantage of measuring the fluorescence in each well simultaneouslythus avoiding the imprecision of sequential measurement well by wellmeasurement. The system is also designed to read the fluorescent signalfrom each well of a 96 or 384 well plate as fast as twice a second. Thisfeature provides FLIPR with the capability of making very fastmeasurements in parallel. This property allows for the measurement ofchanges in many physiological properties of cells that can be used assurrogated markers to a set of functional assays for drug discovery.FLIPR is also designed to have state of the art sensitivity. This allowsit to measure very small changes with great precision.

INFORMATION DISCLOSURE

U.S. Pat. No. 6,022,704, Feb. 8, 2000, DNA and mRNA encoding an alpha 4subunit of human neuronal nicotinic acetylcholine receptor and cellstransformed with same, Elliott, K. J. et. al.

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BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Construction of the α7/5-HT₃ Chimeric Ligand Gated Ion Channel

FIG. 2 Amino Acid Sequence of the mature cell surface form of theα7/5-HT₃ Chimeric Ligand Gated Ion Channel. (mutant α7 receptors of SEQID NOS: 10, 12, 14) have same mature amino terminus)Underlined=N-terminal AA (1-201)from human α7 nAChR gene Notunderlined=C-terminal AA from mouse 5-HT₃ gene Bold font=position oftransmembrane domain 1

FIG. 3 Fl-btx binding to the α7/5-HT₃ Chimeric Ligand Gated Ion Channel

FIG. 4 Epibatidine Competes Fl-btx Binding to α7/5-HT₃ Chimeric LigandGated Ion Channel

FIG. 5 α-btx Competes F1-btx Binding to α7/5-HT₃ Chimeric Ligand GatedIon Channel

FIG. 6 Non-Physiologic Buffer Increases Calcium Flux through theα7/5-HT₃ Chimeric Ligand Gated Ion Channel

FIG. 7 Non-Physiologic Buffer does not Increase the Bradykinin-InducedCalcium Flux

FIG. 8 Exemplary Data from a screen for modulators of activityindicating a test compound is an antagonist

FIG. 9 Assay of function of double mutant human α7 ligand gated ionchannel

FIG. 10 Exemplary Data from a screen for modulators of activityindicating a test compound is an antagonist

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Sequence 1 DNA coding sequence of the wild type human α7 ligand gatedion channel

Sequence 2 Amino acid sequence of the wild type human α7 ligand gatedion channel

Sequence 3 DNA coding sequence of the murine 5HT₃ ligand gated ionchannel

Sequence 4 Amino acid sequence of the murine 5HT₃ ligand gated ionchannel

Sequence 5 DNA coding sequence of the human α7/murine 5HT₃ ligand gatedion channel

Sequence 6 Amino acid sequence of the human α7/murine 5HT₃ ligand gatedion channel

Sequence 7 GG443 PCR Primer

Sequence 8 GG444 PCR Primer

Sequence 9 DNA coding sequence of the mutant human α7 ligand gated ionchannel containing the T→P mutation at amino acid position 230

Sequence 10 Amino acid sequence of the mutant human α7 ligand gated ionchannel containing the T→P mutation at amino acid position 230

Sequence 11 DNA coding sequence of the mutant human α7 ligand gated ionchannel containing the C→S mutation at amino acid position 241

Sequence 12 Amino acid sequence of the mutant human α7 ligand gated ionchannel containing the C→S mutation at amino acid position 241

Sequence 13 DNA coding sequence of the double mutant human α7 ligandgated ion channel containing the T→P mutation at amino acid position 230and the C→S mutation at amino acid position 241

Sequence 14 Amino acid sequence of the double mutant human α7 ligandgated ion channel containing the T→P mutation at amino acid position 230and the C→S mutation at amino acid position 241

SUMMARY OF THE INVENTION

The present invention addresses the need identified above in that itprovides methods and compositions useful for inducing inward conductingcation channels and cell lines expressing said channels topreferentially conduct calcium. Said inward cation channels can beeither voltage gated ion channels, ligand gated channels, or non-voltagenon-ligand gated ion channels.

In one embodiment, the invention includes a special cell culture mediumcomprising a high concentration of calcium and a relatively lowconcentration of sodium. The special cell culture medium comprisescalcium ions at a concentration of from about 2 to 10 mM, sodium ions ata concentration of from about 0 to 50 mM, a pH between about 7.0-7.5,potassium between about 0.1-30 mM and a buffer compatible with mammaliancells. Because the ionic composition of the medium is reduced by thereduction in sodium ion content typically supplied by isotonicconcentrations of sodium chloride the isotonicity of the media isretained by the addition of an impermeant cation in an amount sufficientto maintain isotonic conditions.

In another embodiment the invention includes methods of treating cellsin aqueous culture medium, where the treatment comprises changing theaqueous environment of the cells from their beginning state, where theymay exist in any aqueous buffered solution designed to maintain livingcells, to a special cell culture medium where the ionic conditionscomprise: calcium ions at a concentration of from about 2 to 10 mM,sodium ions from about 0 to 50 mM, pH from about 7.0 to 7.5 andimpermeant cations in an amount sufficient to maintain isotonicconditions.

In another embodiment the invention includes methods of inducing cellsthat express either voltage gated, ligand gated or non-voltagenon-ligand gated inward conducting cation channels to preferentiallyconduct calcium ions. This is known as calcium conductance or calciumflux, comprising: incubating the cells in a special cell culture mediumdescribed above for a length of time from between 15 minutes to about 8hours. The conductance can then be measured in a variety of ways. A fewof which are described.

In another particularly preferred embodiment the invention includesmethods of inducing cells that express α7/5HT₃ chimeric receptors topreferentially conduct calcium ions comprising the step of incubatingthe cells in the above mentioned special cell culture media.

In another particularly preferred embodiment the invention includesmethods of inducing cells that express a mutant α7 receptor topreferentially conduct calcium ions comprising the step of incubatingthe cells in the above mentioned special cell culture media.

In another embodiment the invention provides a chimeric α7/5-HT₃ nucleicacid molecule encoding a heretofore unknown chimeric ligand gated ionchannel and constructs and recombinant host cells incorporating theisolated nucleic acid molecules; chimeric α7/5-HT₃ polypeptides encodedby the isolated nucleic acid molecule and methods of making and usingall of the foregoing.

In yet another embodiment the invention provides heretofore unknownmutants of the human α7 nAChR ligand gated ion channel and constructsand recombinant host cells incorporating the isolated nucleic acidmolecules; mutant α7 nAChR polypeptides encoded by the isolated nucleicacid molecules and methods of making and using all of the foregoing.

SEQ ID NOS: 5, 6, 9, 10, 11, 12, 13 and 14 provides particularhuman/mouse chimeric polynucleotide and polypeptide sequences and mutantα7 nAChR polynucleotide and polypetide sequences, and the invention isincludes within its scope other human and mouse allelic variants andconservative amino acid substitutions. The polynucleotide sequences areintended to encompass the well known degeneracy of the genetic code.

In yet other embodiment the invention provides a fluorescent ligandbinding assay comprising: incubating cells with a fluorescent ligandcapable of binding to cell surface receptors and measuring thefluorescence of cell bound ligand using FLIPR. The invention alsodescribes assays for selective agonists, antagonists and modulators ofthe α7 nAChR.

ADDITIONAL DETAILS OF THE INVENTION

There are many calcium influx assays suitable for high throughputscreening but there are no good high throughput assays to measure theinflux of other cations. Therefore it is desirable to induce a cell linethat expresses inward conducting cation channels normally conductingother cations to preferentially conduct calcium. The present inventionprovides a methods and compositions of adapting an inward conductingcation channel to preferentially conduct calcium. Such inward conductingcation channels include voltage gated ion channels, ligand gated ionicchannels, and non-voltage gated non-ligand gated ionic channels. Voltagegated ionic channels may be described as ion channels which open inresponse to a change in the voltage across the membrane. Ligand gatedion channels may be described as ion channels which open in response tothe binding of a ligand to the channel protein. Non-voltage non-ligandgated ion channels may be described as channels which don not open inresponse to either voltage across the membrane or to ligand binding butthat are regulated by covalent modifications by second messengersignaling pathways such as protein phosphorylation, or increases inchannel gene expression leading to increases in ion channel density.Such a condition may exist, for example, in epithelial cells such askidney epithelium cells and white blood cells.

As used herein the term “5HT-3 receptor” is used interchangeably with“5HT ligand gated ion channel” As used herein the term “α7 receptor” and“α7 nAChR” and “α7 ligand gated ion channel” are all usedinterchangeably. The term “mutant α7 receptors”, “mutant α7 ligand gatedion channel” or mutant “α7 AchR” refers any one of a number of specificmutant polynucleotide or polypeptide species described herein. When aspecific mutation is desired it referred to by the SEQ ID NO of itsencoding nucleic acid, or by reference to the SEQ ID NO of the resultantpredicted polypeptide product. By way of example, a cell line expressinga particular mutation might be referred to as cells expressing thepolynucleotide sequence of SEQ ID NO: 13 or the polypeptide sequence ofSEQ ID NO:14. As aid in understanding the reader is directed to thesection entitled “Brief Description of the Sequence Listings”

Special Cell Culture Medium

The inventors provide an ionic environment that can be used with all ofthe ion channels described herein. The special cell culture mediumprovides a means of adapting ligand gated, voltage gated, and non-ligandgated non-voltage gated ion channels not normally conducting calcium tothe conductance of calcium. The special cell culture medium provides ameans of adapting those channels normally conducting sodium, potassiumor other ions to the conductance of calcium whether those channels be ofthe ligand gated, voltagen gated, or non-ligand non voltage gatedvariety.

The inventors have addressed the task of inducing calcium flux orcalcium conductance or transmission of calcium ions in ion channels notnormally preferentially transmitting calcium ions by providing specialcell culture compositions comprising a high concentration of calcium anda relatively low concentration of sodium. The special cell culturemedium comprises calcium ions at a concentration of from about 2 to 10mM, sodium ions at a concentration of from about 0 to 50 mM, a pHbetween about 7.0-7.5, potassium between about 0.1-30 mM and a buffercompatible with mammalian cells. It is understood by one of skill in theart that a variety of salts may be used as a source of sodium ionsincluding but not limited by the examples of NaCl, Na2HPO4, NaH2PO4 andNaHCO3. It is understood by one of skill in the art that a variety ofsalts may be used as a source of potassium ions including but notlimited by the examples of KCl, K2HPO4, KH2PO4 and KHCO3. It isunderstood by one of skill in the art that calcium ions may be suppliedby a variety of salts including but not limited by the examples of CaCl2and CaSO4. In addition all of the above ions may be supplied by salts oforganic compounds within the knowledge of one of skill in the art.

Because the ionic composition of the medium is reduced by the reductionin sodium ion content typically supplied by isotonic concentrations ofsodium chloride the isotonicity of the media is retained by the additionof an impermeant cation in an amount sufficient to maintain isotonicconditions. In the context of the present invention, the term “isotonic”means having an osmolality that is within the range tolerated by thecell or a solution that has the same osmotic pressure as the interior ofthe cell. Usually this is in the range of about 285-315 mOsm/kg H2Odepending on the cell type and source, more preferably about 290-305,for most cell types this is about 300 mOsm/kg H2O.

Impermeant cations are defined as organic cations too large to passthrough the channel of interest. By way of example only, such cationsmay include N-methyl-D-glucamine, choline, tetraethylammonium (TEA),tetrethymethyammonium (TMA) and tetrapropylammonium (TPA) and Tris.

In one particular embodiment, the cell culture medium comprises CaCl₂ atabout 4 mM, MgSO₄ at about 0.8 mM, HEPES Buffer at about 20 mM, Glucoseat about 6 mM, NaCl at about 20 mM, KCl at about 5 mM and the impermeantcation N-methyl-D-glucamine at about 120 mM.

It is understood by one skilled in the art that calcium flux or thetransmission of calcium ions may be accessed by a number of well knowmethods. These include but are not limited by the measurement of voltagechanges either directly or indirectly caused by the movement of calciumions ie. measuring ionic flux or conductance. In addition the presenceof calcium may be accessed by its interaction with a number offlourescent dyes well known in the art. These include but are notlimited by the choices of Calcium Green and flou-3 and flou-4. It isunderstood that the fluorescent signal of the various dyes known in theart may be measured on FLIPR but also on other more conventionalinstrumentation including fluorimeters

The present invention also provides a α7/5-HT₃ chimeric receptor and anovel mutant human α7 receptors encoded by isolated polynucleotides(e.g., DNA sequences and RNA transcripts, both sense and complementaryantisense strands, both single and double-stranded, including splicevariants thereof) encoding a human enzyme referred to herein as α7/5-HT₃chimera or mutant α7 receptor DNA. Polynucleotides of the inventioninclude cDNA, and DNA that has been chemically synthesized in whole orin part. “Synthesized” as used herein and understood in the art, refersto polynucleotides produced by purely chemical, as opposed to enzymatic,methods. “Wholly” synthesized DNA sequences are therefore producedentirely by chemical means, and “partially” synthesized DNAs embracethose wherein only portions of the resulting DNA were produced bychemical means. “Isolated” as used herein and as understood in the art,whether referring to “isolated” polynucleotides or polypeptides, istaken to mean that it is uniquely created by the inventors, separatedfrom the original cellular or genetic environment in which thepolypeptide or nucleic acid is normally found. As used herein therefore,by way of example only, a transgenic animal or a recombinant cell lineconstructed with a polynucleotide of the invention, incorporates the“isolated” nucleic acid.

Allelic variants are modified forms of a wild type gene sequence, themodification resulting from recombination during chromosomal segregationor exposure to conditions which give rise to genetic mutation. Allelicvariants, like wild type genes, are naturally occurring sequences (asopposed to non-naturally occurring variants which arise from in vitromanipulation).

A DNA sequence encoding a α7/5-HT₃ polypeptide is set out in SEQ ID NO:5. DNA sequences encoding the mutant α7 receptor polypeptides are setout in SEQ ID NO: 9, 11 and 13. One of skill in the art will readilyappreciate that the preferred DNA of the invention comprises a doublestranded molecule, for example the molecule having the sequence setforth in SEQ ID NO: 5, 9, 11 or 13 along with the complementary molecule(the “non-coding strand” or “complement”) having a sequence deduciblefrom the sequence of SEQ ID NO: 5, 9, 11, or 13 according toWatson-Crick base pairing rules for DNA. Also preferred are otherpolynucleotides encoding the α7/5-HT₃ polypeptides or mutantpolypeptides of SEQ ID NO: 6, 10, 12, or 14 which differ in sequencefrom the polynucleotides of SEQ ID NO: 5, 9, 11 or 13 by virtue of thewell known degeneracy of the genetic code.

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art.

Autonomously replicating recombinant expression constructs such asplasmid and viral DNA vectors incorporating polynucleotides of theinvention are also provided. Expression constructs wherein α7/5-HT₃chimera receptor or the novel mutant human α7 receptor-encodingpolynucleotides are operatively linked to an endogenous or exogenousexpression control DNA sequence and a transcription terminator are alsoprovided. Expression control DNA sequences include promoters, enhancers,and operators, and are generally selected based on the expressionsystems in which the expression construct is to be utilized. Preferredpromoter and enhancer sequences are generally selected for the abilityto increase gene expression, while operator sequences are generallyselected for the ability to regulate gene expression. Expressionconstructs of the invention may also include sequences encoding one ormore selectable markers that permit identification of host cells bearingthe construct. Expression constructs may also include sequences thatfacilitate, and preferably promote, homologous recombination in a hostcell. Preferred constructs of the invention also include sequencesnecessary for replication in a host cell.

Expression constructs are preferably utilized for production of anencoded protein, but also may be utilized simply to amplify a α7/5-HT₃chimera receptor or the novel mutant human α7 receptor -encodingpolynucleotide sequence.

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, comprising a polynucleotideof the invention (or vector of the invention) in a manner which permitsexpression of the encoded α7/5-HT₃ chimera receptor or the novel mutanthuman α7 receptor polypeptide. Polynucleotides of the invention may beintroduced into the host cell as part of a circular plasmid, or aslinear DNA comprising an isolated protein coding region or a viralvector. Methods for introducing DNA into the host cell well known androutinely practiced in the art include transformation, transfection,electroporation, nuclear injection, or fusion with carriers such asliposomes, micelles, ghost cells, and protoplasts. Expression systems ofthe invention include bacterial, yeast, fungal, plant, insect,invertebrate, and mammalian cells systems.

Host cells for expression of α7/5-HT₃ chimera receptor or the novelmutant human α7 receptor polypeptides include prokaryotes, yeast, andhigher eukaryotic cells. Suitable prokaryotic hosts to be used for theexpression of α7/5-HT₃ chimera receptor and or a mutant α7 receptorsinclude but are not limited to bacteria of the genera Escherichia,Bacillus, and Salmonella, as well as members of the genera Pseudomonas,Streptomyces, and Staphylococcus.

The isolated nucleic acid molecules of the invention are preferablycloned into a vector designed for expression in eukaryotic cells, ratherthan into a vector designed for expression in prokaryotic cells.Eukaryotic cells are preferred for expression of genes obtained fromhigher eukaryotes because the signals for synthesis, processing, andsecretion of these proteins are usually recognized, whereas this isoften not true for prokaryotic hosts (Ausubel, et al., ed., in ShortProtocols in Molecular Biology, 2nd edition, John Wiley & Sons,publishers, pg.16-49, 1992.). Eukaryotic hosts may include, but are notlimited to, the following: insect cells, African green monkey kidneycells (COS cells), Chinese hamster ovary cells (CHO cells), human 293cells, human SH-EP1 cells and murine 3T3 fibroblasts.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker genes. Such genes generally encode,e.g., a protein that confers antibiotic resistance or that supplies anauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescriptvectors (Stratagene), and pQE vectors (Qiagen).

The α7/5-HT₃ chimera receptor and the novel mutant human α7 receptor mayalso be expressed in yeast host cells from genera includingSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Yeast vectors will often contain an originof replication sequence from a 2 micron yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Vectors replicable in both yeast and E. coli(termed shuttle vectors) may also be used. In addition to theabove-mentioned features of yeast vectors, a shuttle vector will alsoinclude sequences for replication and selection in E. coli.

Insect host cell culture systems may also be used for the expression ofhuman α7/5-HT₃ chimera receptor or the novel mutant human α7 receptor IIpolypeptides. In a preferred embodiment, the α7/5-HT₃ chimera receptorand the novel mutant human α7 receptor II polypeptides of the inventionare expressed using a baculovirus expression system. Further informationregarding the use of baculovirus systems for the expression ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988).

In another preferred embodiment, the α7/5-HT₃ chimera receptor or thenovel mutant human α7 receptor II polypeptide is expressed in mammalianhost cells. Non-limiting examples of suitable mammalian cell linesinclude the COS-7 line of monkey kidney cells (Gluzman et al., Cell23:175 (1981)), Chinese hamster ovary (CHO) cells, and human 293 cells.

The choice of a suitable expression vector for expression of the humanα7/5-HT₃ chimera receptor or the novel mutant human α7 receptor IIpolypeptid of the invention will of course depend upon the specific hostcell to be used, and is within the skill of the ordinary artisan.Examples of suitable expression vectors include pcDNA3 (Invitrogen) andpSVL (Pharmacia Biotech). Expression vectors for use in mammalian hostcells may include transcriptional and translational control sequencesderived from viral genomes. Commonly used promoter sequences andenhancer sequences which may be used in the present invention include,but are not limited to, those derived from human cytomegalovirus (CMV),Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for theconstruction of mammalian expression vectors are disclosed, for example,in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol.Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984));EP-A-0367566; and WO 91/18982.

The invention also provides α7/5-HT₃ chimera receptor or novel mutanthuman α7 receptor II polypeptides encoded by a polynucleotides of theinvention. Presently preferred is α7/5-HT₃ chimera polypeptidecomprising the amino acid sequence set out in SEQ ID NO: 6 and a novelmutant human α7 receptor comprising the amino acid sequence set out inSEQ ID NO: 14

Polypeptides of the invention may be produced natural cell sources ormay be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Glycosylated andnon-glycosylated form of α7/5-HT₃ chimera receptor or the novel mutanthuman α7 receptor II are embraced.

The invention also embraces variant α7/5-HT₃ chimera receptor or thenovel mutant human α7 receptor polypeptides wherein the essentialactivity, including pharmacology which accurately mimics that of thenative α7 ligand gated ion channel receptor of the α7/5-HT₃ chimerareceptor or the novel mutant human α7 receptor II is maintained.Examples of such variants include insertion, deletions or substitutions.Insertional variants also include fusion proteins wherein the aminoand/or carboxy termini of the α7/5-HT₃ chimera receptor or the novelmutant human α7 receptor is fused to another polypeptide. It is furtherenvisioned that the although the polypeptides of the invention aredisclosed as mature protein sequences in SEQ ID NOS: 6, 10, 12, and 14,which include a signal sequence necessary for insertion into the cellmembrane, the invention also includes polypeptides with the signalsequence removed. FIG. 2 provides a sequence representing indicatingthat the mature protein of α7 AChR derived polypeptides including themutant polypeptides and the chimeric polypeptide have 22 amino acidsremoved in the mature form.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a α7/5-HT₃ chimera receptor or the novelmutant human α7 receptor polypeptide are removed. Deletions can beeffected at one or both termini of the α7/5-HT₃ chimera receptor or thenovel mutant human α7 receptor polypeptide, or with removal of one ormore residues within the α7/5-HT₃ chimera receptor or the novel mutanthuman α7 receptor amino acid sequence.

In still another aspect, the invention provides substitution variants ofα7/5-HT₃ chimera receptor and the novel mutant human α7 receptorpolypeptides. Substitution variants include those polypeptides whereinone or more amino acid residues of a α7/5-HT₃ chimera receptor and thenovel mutant human α7 receptor polypeptide are removed and replaced withalternative residues. In one aspect, the substitutions are conservativein nature, however, the invention embraces substitutions that are alsonon-conservative. Conservative substitutions for this purpose may bedefined as set out in Tables A, B, or C below.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable A (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-chargedDE KR Aromatic HFWY Other NQDE

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp.71-77] as set out in Table B, immediately below

TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: STYB. Amides: NQ C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): KRH Negatively Charged (Acidic): DE

As still an another alternative, exemplary conservative substitutionsare set out in Table C, immediately below.

TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

EXAMPLE 1 Construction of Chimeric α7/5-HT₃ Receptor

PCR Primers GG443 (SEQ ID NO:7) and GG444 SEQ ID NO:8 were used toisolate the DNA encoding the N-terminal 201 amino acids from the humanα7 nAChR (FIG. 1).

GG443: 5′GGCTCTAGACCACCATGCGCTGTTCACCGGGAGGCGTCTGGCTG 3′

GG444: 5′ GGGTGATCACTGTGAAGGTGACATCAGGGTAGGGCTC 3′

The isolated DNA fragment of encoding the N-terminus of the α7 wasengineered to have an Xba 1 site at the 5′ end and Bcl 1 site at the 3′end. The engineered restriction sites are underlined in each respectiveprimer. The pore forming domain of the mouse 5-HT₃ cDNA was thenisolated as a Bcl 1/Sal 1 DNA fragment of the complete mouse cDNA gene.A ligation reaction was used to join the 5′ of the α7 cDNA with the 3′end of the 5-HT₃ cDNA. This ligated fragment was isolated and purifiedand then cloned into the Xba1 Sal 1 site of two mammalian expressionplasmid vectors termed pGG764 and pGG759. The parental plasmid termedpGG764, which contained the G418 resistance gene also contained acytomegalovirus (CMV) promoter and a bovine growth hormonepolyadenylation site for the initiation and termination of mRNAtranscription. The parental plasmid termed pGG759 contained thehygromycin resistance gene and the identical mRNA initiation andtermination regulatory elements. The new plasmid derived from theinsertion of α7/5-HT₃ gene into pGG764 was termed pGS 175. The newplasmid derived from the insertion of α7/5-HT₃ gene into pGG759 wastermed pGS176. Both pGS175 and pGS179 were transformed into E. coli andisolated colonies were picked and expanded. The DNA from each plasmidwas isolated and sequenced to verify that both constructions werecorrect. The sequence obtained for the coding region of the α7/5-HT₃cDNA construct is shown in SEQ ID NO: 5 and the predicted amino acidsequence of the construct is given in in SEQ ID NO: 6

It is understood that once one skilled in the art has possession ofapplicant's chimeric α7/5-HT₃ and mutant α7 AChRs a number of novelassays are evident for the assessment of ligand binding, of the abilityof test compounds to function as agonists, and to measure the ability oftest compounds to function as modulators of α7 activity. Details areprovided in the examples below. It is understood however that oneskilled in the art might perform the same essential functions in avariety of way and the examples are in no way intended to indicatelimitations in the claims.

Expression of the Chimeric Receptor

The α7/5-HT₃ cDNA inserted into pGS175 and pGS179 were simultaneouslytransfected into SH-EP1 cells using cationic lipid transfection reagentand cells expressing the α7/5-HT₃ channel were selected using 800 μg/mlgeneticin (G418) and 400 μg/ml of hygromycin B. Cells expressing thechimeric protein at high levels were identified by measuringfluorescein-α-bungarotoxin binding (see FIG. 3). Isolated clones weregrown in Eagle's minimal essential medium (MEM) supplemented with 10%fetal bovine serum (FBS), 4 mM L-Glutamine, Fungi-Bact.(1:100), 400μg/ml hygromycin B, and 800 μg/ml G418. All cells were maintained in anincubator at 37° C. in a humidified 6% CO₂ atmosphere.

EXAMPLE 2 Fluorescein Labeled α-bungarotoxin (fl-btx) Binding Assay

The α7/5-HT₃-SHEP cells were grown in minimal essential medium (MEM)containing nonessential amino acids supplemented with 10% fetal bovineserum, L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/mlfungizone, 400 μg/ml Hygromycin-B, and 800 μg/ml Geneticin. The cellswere grown in a 37° C. incubator with 6% CO₂. The α7/5-HT₃-SHEP cellswere trypsinized and plated in 96 well plates with dark side walls andclear bottoms (Corning #3614) at density of 26×10⁴ cells per well twodays before analysis. On the day of the analysis, the cells were washfour times using a Bio-Tek plate washer. After the fourth cycle, thefinal volume in each well was100 μl. Cellular fluorescence was analyzedon FLIPR (Molecular Devices) after the addition of a 100 μl of a 2×stockfluorescein labeled α-bungarotoxin (F-1176 Molecular Probes: Fl-btx). Incompetition experiments the competing ligand was added as a 2×drug stockbefore the addition of Fl-btx. Fluorescence was measured by exciting thedye at 488 nm using 500 mW of power. A 0.5 second exposure was used toilluminate each well. Fluorescence emmission was recorded above 525 nm.Fluorescence was detected using a F-stop set of either 2.0 or 1.2. Thecellular fluorescence was so intense that subsequent washing was notneeded to measure cellular fluorescence.

The data in FIG. 3 shows that Fl-btx binding is a saturable reactionwith a Ki of 15.5 nM. Nicotine at 100 μM competes at all concentrationsof Fl-btx (FIG. 3). FIGS. 4 and 5 show that epibatidine and unlabeledα-btx also compete for Fl-btx binding with a Ki of 90 nM and 33 nMrespectively. The data in Table 1 provide a summary of the effect ofseven structurally unrelated molecules in the whole cell Fl-btx bindingassay.

Agonists/Antagonists Fitc-α-Bungoarotoxin Binding (30 nM) (−) NicotineIC₅₀ = 9.7 μM (+/−) Epibatidine IC₅₀ = 90 nM GTS-21 IC₅₀ = 16 μM ABT-418IC₅₀ = 38 μM Anabasiene IC₅₀ = ND Mecamylamine IC₅₀ = >300 μMMethyllcaconitine (MLA) IC₅₀ = 26 nM

The rank order potency of these compounds follow the known pharmacologyof α7 nAChR (Holliday et al 1997). Taken together these data show thatthe fl-btx binding assay on the α7/5-HT₃ chimera receptor can be used tonovel and selective agonists and antagonists of endogenous α7 nAChR.

The whole cell binding assay described in this example is useful in manyregards not the least of which is that α7 nAChR is in its nativeconfiguration, only cell surface α7 nAChR is a binding target, the assayis simpler because there is no need to prepare membranes, and there areno radioisotopes being used and because fluorescence is detected withinapproximately 200 microns of the bottoms of the wells the need forextensive washing is eliminated.

Our results as summarized in the Figures demonstrate that the α7/5-HT₃SH-EP cell line can be used in the Fl-btx binding assay on FLIPR. Thepharmacology of the α7/5-HT₃ receptor suggests that the Fl-btx bindingassay can be used in a HTS format to find novel α7 nAChR agonists andantagonists.

EXAMPLE 3 Calcium Flux Assay—Identification of an α7 nAChR Agonist

The α7/5-HT₃-SHEP or alternatively the human α7 nACHR double mutant SHEP(described below) cells were grown in minimal essential medium (MEM)containing nonessential amino acids supplemented with 10% fetal bovineserum, L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/mlfungizone, 400 μg/ml Hygromycin-B, and 800 μg/ml Geneticin. The cellswere grown in a 37° C. incubator with 6% CO₂. The α7/5-HT₃-SHEP cellswere trypsinized and plated in 96 well plates with dark side walls andclear bottoms (Corning #3614) at density of 26×10⁴ cells per well twodays before analysis. The cells were loaded in a 1:1 mixture of 2 mMCalcium Green-1, AM (Molecular Probes) prepared in anhydrousdimethylsulfoxide and 20% pluonic F-127 (Molecular Probes). This reagentwas added directly to the growth medium of each well to achieve a finalconcentration of 2 μM of Calcium Green-1, AM. The cells were incubatedin the dye for one hour at 37° C. and then washed with 4 cycles ofBio-Tek plate washer. Each cycle was programmed to wash each well withfour times with either EBSS or MMEBSS. After the third cycle, the cellswere allowed to incubate at 37° C. for at least ten minutes. After thefourth cycle final volume in each well was 100 μl. The cells wereanalyzed on FLIPR (Molecular Devices) for the change in fluorescenceafter the addition of a 100 μg of a 2×drug stock. FLIPR was set up toexcite the dye with at 488 nanometers using 500 mW of power. A 0.5second exposure was used to illuminate each well. Fluorescence emissionwas recorded above 525 nm. Fluorescence was detected using a F-stop setof either 2.0 or 1.2.

Under physiological ionic conditions, the 5-HT₃ ligand gated ion channelconducts primarily Na⁺ and is a poor conductor of Ca⁺⁺(Yang 1990; Brownet al 1998). Whereas, under physiological ionic conditions the α7 nAChchannel conducts primarily Ca⁺⁺.

Therefore a particular embodiment of a special cell culture media,designated MMEBSS was used to enhance the agonist-evoked flux of calciumthrough the α7/5-HT₃ channel expressed in SH-EP1 cells (FIG. 6). Wecompared the physiological Earles Balanced Salt Solution (EBBS) bufferand the special cell culture media (MMEBSS) in the Ca⁺⁺ functional assayon FLIPR. The result of this experiment clearly indicated that underphysiological conditions (EBBS) little calcium was detected in responseto a maximally effective concentration of (−) nicotine (100 μM). Otherthe other hand using the special cell culture media, (MMEBSS) 100 μM (−)nicotine evoked a large increase in intracellular calcium (FIG. 6).Under these conditions, FLIPR can be used to accurately measure agonistactivity of the α7/5-HT₃ channel (Table 2). The α7/5-HT₃-SH-EP1 cellsexpress an endogenous bradykinin receptor that when stimulated with 100nM bradykinin produces a maximal increase in intracellular calcium byreleasing calcium from intracellular stores. The data in FIG. 7 showthat the bradykinin-induced calcium flux was similar in EBSS and MMEBSS.These data indicate that the effect of MMEBSS was specific for thecalcium flux through the α7/5-HT3 channel

The special cell culture media, designated MMEBSS is comprised of 4 mMCaCl₂, 0.8 mM MgSO₄, 20 mM NaCl, 5.3 mM KCL, 5.6 mM D-Glucose, 120 mMN-Methyl-D-Glucosamine, 9 mM Tris base and 20 mM HEPES. A detaileddescription of the preparation of MMEBSS is provided below. It should berecognized however that the recipe below is provided by way of exampleonly and that the applicants intends to claim the full range of whatthey have invented.

MMEBSS Buffer Stock Buffer Component Solution 2 Liters FinalConcentration CaCl₂ Dihydrate 1 M 10 ml. 4 mM MgSO₄7H₂O 1 M 1.6 ml. 0.8mM NaCl 2 M 20 ml. 20 mM KCl 0.8 gr. 5.3 mM D-Glucose 2.0 gr. 5.6 mMTris-HEPES¹ 1 M 40 ml. 20 mM N-Methyl-D-Glucamine 1.36 176.5 ml 120 mM(pH 7.3)² Tris Base³ 0.5 gr ¹1 M. Tris-HEPES pH 7.4 is formulated byweighing 47.66 grams of HEPES and adding approximately 8 of Tris base in150 ml of water, the pH is adjusted to 7.4 with HCl. The final volume isadjusted to 200 ml. ²1.36 M. N-Methyl-D-Glucamine/HCl pH 7.3 isformulated by adding 265.47 grams of N-Methyl-D-Glucamine in 500 ml.water 115 ml concentrated HCl is then added to the solution withstirring. The final pH is adjusted to 7.4 ³Final concentration of Trisin buffer is approximately 9 mM

For the experiments described above the physiologic buffer designatedEarles Balanced Salt Solution was also prepared or purchased.

The compositions of EBSS and MMEBSS are compared below.

Earle's Balanced Salt Solution (EBSS) CaCl₂ 1.8 mM MgSO₄ 0.8 nM NaHPO₄1.0 mM NaHCO₃ 26 mM Hepes 20 mM Glucose 5.6 mM NaCl 117.0 mM KCl 5.3 mMMMEBSS CaCl₂ 4.0 mM MgSO₄ 0.8 mM NaHPO₄ 0.0 mM NaHCO₃ 0.0 mM Hepes 20 mMGlucose 5.6 mM NaCl 20.0 mM KCl 5.3 mM N-methyl-D- 120 mM glucamine alsoincludes Tris base

The summary of the pharmacological results using the α7/5-HT₃ channel asa drug target is listed in Table 2.

Characterization of the α7/5-HT₃ Chimeric Channel α7/5-HT₃ α7 nAChR EC₅₀(μM)* EC₅₀ (μM)* Nicotine 5.7 10-50 Epibatidine 0.120  2 ABT 27 70Anabaseine 6.6  6 GTS-21 30 30 *“EC₅₀” is the effective concentrationthat produces a 50% maximal response.

These data establish that agonist activity of the α7/5-HT₃ channel canbe used to predict the agonist activity at the endogenous α7 nAChreceptor and thus provide evidence that the α7/5-HT₃ channel can be useas a drug target to find novel α7 nAChR agonists.

EXAMPLE 4 Calcium Flux Assay—Identification of an α7 nAChR Antagonist

The SH-EP1 cells expressing the 7/5-HT₃ nACHR (7/5-HT₃-SHEP) oralternatively the human α7 nACHR double mutant SHEP cells(describedbelow) were grown in minimal essential medium (MEM) containingnonessential amino acids supplemented with 10% fetal bovine serum,L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/ml fungizone,400 ug/ml Hygromycin-B, and 800 ug/ml Geneticin. The cells were grown ina 37° C. incubator with 6% CO₂. The 7/5-HT₃-SHEP cells were trypsinizedand plated in 96 well plates with dark side walls and clear bottoms(Corning #3614) at density of 26×10⁴ cells per well two days beforeanalysis. The 7/5-HT₃-SHEP cells were loaded in a 1:1 mixture of 2 mMCalcium Green-1, AM (Molecular Probes) prepared in anhydrousdimethylsulfoxide and 20% pluonic F-127 (Molecular Probes). This reagentwas added directly to the growth medium of each well to achieve a finalconcentration of 2 M of Calcium Green-1, AM. The α7/5-HT₃-SHEP cellswere incubated in the dye for one hour at 37° C. and then washed with 4cycles of Bio-Tek plate washer. Each cycle was programmed to wash eachwell with four times with either EBSS or MMEBSS. After the third cycle,the α7/5-HT₃-SHEP cells were allowed to incubate at 37° C. for at leastten minutes. After the fourth cycle final volume in each well was100 l.Antagonist activity was measured as a decrease in nicotine-inducedcalcium influx using α7/5-HT₃ channel as a drug target. FLIPR (MolecularDevices) was set up to measure intracellular calcium by exciting theCalcium Green with at 488 nanometer using 500 mW of power and readingfluorescence emission above 525 nanometers. A 0.5 second exposure wasused to illuminate each well. Fluorescence was detected using a F-stopset of either 2.0 or 1.2. Specifically, after 30 seconds of baselinerecording, test compounds were added to each well of a 96 well plateusing 50 ul from a 3×drug stock. 180 seconds after the addition of thetest compounds, nicotine was added to each well to achieve a finalconcentration of u100 M. In each experiment, 4 wells were used assolvent controls. As indicated in FIG. 8 antagonist activity wasmeasured as a decrease in the 100 M nicotine-induced calcium influxrelative to the effect of u100 M nicotine in the solvent control wells.

EXAMPLE 5 Construction of the Human α7 Mutant Receptors

We discovered that it was possible by introducing certainnon-conservative amino acid changes at the amino acid positionscorresponding to positions 230 and 241 of the human sequence to recreatethe desireable properties of the human/mouse α7nAChR/5-HT₃ hybrid.

The two primer system utilized in the Transformer Site-DirectedMutagenesis kit from Clontech (LaJolla Calif.), may be employed forintroducing site-directed mutants into the human α7 sequence of SEQ IDNO:1 Following denaturation of the target plasmid in this system, twoprimers are simultaneously annealed to the plasmid; one of these primerscontains the desired site-directed mutation, the other contains amutation at another point in the plasmid resulting in elimination of arestriction site. Second strand synthesis is then carried out, tightlylinking these two mutations, and the resulting plasmids are transformedinto a mutS strain of E. coli. Plasmid DNA is isolated from thetransformed bacteria, restricted with the relevant restriction enzyme(thereby linearizing the unmutated plasmids), and then retransformedinto E. coli. This system allows for generation of mutations directly inan expression plasmid, without the necessity of subcloning or generationof single-stranded phagemids. The tight linkage of the two mutations andthe subsequent linearization of unmutated plasmids results in highmutation efficiency and allows minimal screening. Following synthesis ofthe initial restriction site primer, this method requires the use ofonly one new primer type per mutation site. Transformants can bescreened by sequencing the plasmid DNA through the mutagenized region toidentify and sort mutant clones. Each mutant DNA can then be fullysequenced or restricted and analyzed by electrophoresis on MutationDetection Enhancement gel (J. T. Baker) to confirm that no otheralterations in the sequence have occurred (by band shift comparison tothe unmutagenized control).

A mutant α7 is prepared using Transformer TM site-directed mutagenesiskit, according to the manufacturer's protocol roughly outlined above. Inone mutant, a codon in the channel mRNA is changed from ACG to CCG withthe A at position 688 being changed to a C thus creating a mutantchannel with threonine changed to proline at amino acid position number230. The polynucleotide and predicted amino acid sequence of the entiremutant α7 ligand gated ion channel containing the T→P mutation is setforth in SEQ ID NO: 9 and 10 respectively. In another mutant, a codon inthe channel mRNA is changed from TGT to AGT with the T at position 721being changed to A thus creating a mutant channel with cysteine changedto serine at amino acid position 241. The polynucleotide and predictedamino acid sequence of the entire mutant α7 ligand gated ion channelcontaining the C→S mutation is set forth in SEQ ID NO: 11 and 12respectively. In another mutant, both of the above mentioned mutationsare introduced into the same DNA construct encoding a channel mRNA. Thepolynucleotide and predicted amino acid sequence of the double mutant α7ligand gated ion channel containing the T→P mutation and the C→Smutation is set forth in SEQ ID NO: 13 and 14 respectively.

This double mutant channel protein has been shown to exhibit thedesirable characteristics of the chimeric α7/5-HT₃ ligand gated ionchannel including stability and assay characteristics when expressed inhuman SH-EP1 cells. Exemplary expression methods are described elsewhereand are fully within the ordinary skill of one in the art.

EXAMPLE 6 Functional Results with Double Mutant

The SH-EP1 cells expressing the double mutation of SEQ ID NO: 13 (doublemutant SHEP cells )are grown in minimal essential medium (MEM)containing nonessential amino acids supplemented with 10% fetal bovineserum, L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/mlfungizone, 400 ug/ml Hygromycin-B, and 800 ug/ml Geneticin. The cellsare grown in a 37° C. incubator with 6% CO₂. The 7-double mutant SHEPcells were trypsinized and plated in 96 well plates with dark side wallsand clear bottoms (Corning #3614) at density of 2 6×10⁴ cells per welltwo days before analysis. The double mutant-SHEP cells are loaded in a1:1 mixture of 2 mM Calcium Green-1, AM (Molecular Probes) prepared inanhydrous dimethylsulfoxide and 20% pluonic F-127 (Molecular Probes).This reagent was added directly to the growth medium of each well toachieve a final concentration of 2 M of Calcium Green-1, AM. The doublemutant SHEP cells were incubated in the dye for one hour at 37° C. andthen washed with 4 cycles of Bio-Tek plate washer. Each cycle wasprogrammed to wash each well with four times with either EBSS or MMEBSS.After the third cycle, the double mutant-SHEP cells were allowed toincubate at 37° C. for at least ten minutes. After the fourth cyclefinal volume in each well was100 l. Expression of the mutant α7 receptorwas analyzed by measuring agonist-induced changes in intracellularcalcium accumulation. FLIPR (Molecular Devices) was set up to exciteCalcium Green with at 488 nanometer using 500 mW of power and readingfluorescence emission above 525 nanometers. A 0.5 second exposure wasused to illuminate each well. Fluorescence was detected using a F-stopset of either 2.0 or 1.2. Specifically, after 30 seconds of baselinerecording, test compounds were added to each well of a 96 sell plateusing a 100 l from a 2×drug stock. In each experiment, at least 4 wellscontained 7/5-HT₃-SHEP cells as positive controls. As indicated in FIG.9 agonist activity was measured as an increase in intracellular calciumover baseline. As indicated in FIG. 9 this paradigm identified clonalcell lines that functionally expressed the double mutant α7 receptor.All attempts to express the wild type 7 nAChR using similar methods weretotally unsuccessful.

EXAMPLE 7 Calcium Flux Assay: Modulator Screen

The SH-EP1 cells expressing the double mutation of SEQ ID NO:13 (doublemutant SHEP cells) were grown in minimal essential medium (MEM)containing nonessential amino acids supplemented with 10% fetal bovineserum, L-glutamine, 100 units/ml penicillin/streptomycin, 250 ng/mlfungizone, u400 g/ml Hygromycin-B, and 800 ug/ml Geneticin. The cellswere grown in a 37° C. incubator with 6% CO₂. The cells were trypsinizedand plated in 96 well plates with dark side walls and clear bottoms(Corning #3614) at density of 26×10⁴ cells per well two days beforeanalysis. The double mutant SHEP cells were loaded in a 1:1 mixture of 2mM Calcium Green-1, AM (Molecular Probes) prepared in anhydrousdimethylsulfoxide and 20% pluronic F-127 (Molecular Probes). Thisreagent was added directly to the growth medium of each well to achievea final concentration of 2 M of Calcium Green-1, AM. The double mutantSHEP cells were incubated in the dye for one hour at 37° C. and thenwashed with 4 cycles of Bio-Tek plate washer. Each cycle was programmedto wash each well with four times with either EBSS or MMEBSS. After thethird cycle, the double mutant-SHEP cells were allowed to incubate at37° C. for at least ten minutes. After the fourth cycle final volume ineach well was100 ul. Allosteric modulator activity was measured as thedrug dependent increase in the agonist activity using the double mutantAChR channel as a drug target. Modulator induce increase in agonistactivity was measured by increasing intracellular calcium accumulation.FLIPR (Molecular Devices) was set up to excite Calcium Green with at 488nanometer using 500 mW of power and reading fluorescence emission above525 nanometers. A 0.5 second exposure was used to illuminate each well.Fluorescence was detected using a F-stop set of either 2.0 or 1.2.Specifically, after 30 seconds of baseline recording, test compoundswere added to each well of a 96 well plate using a 50 l from a 3×drugstock. In each experiment, 4 wells were used as solvent controls. Asindicated in FIG. 10 modulator activity produced an increase in thenicotine-induced influx of intracellular calcium. The preferredmodulator had no effect in the absence of agonist. All data is plottedrelative to the effect of 100 M nicotine, which induced a maximalcalcium influx.

EXAMPLE 8

Changing the ionic conditions of cellular medium is also likely toincrease the calcium influx on many other ion channels that do notconduct calcium under physiological conditions. For example, it is knownthat the P2X(2) family of purinoceptors are cation-selective channelsthat are activated by ATP and its analogues. The ionic selectivity ofthis channel is K⁺>Rb⁺>Cs⁺>Na⁺>Li⁺>>>Ca⁺⁺. In addition, divalent ionssuch induce a block of the channel that is measured by a reduction inamplitude of the unitary currents. Organic cations such as NMDG(+),Tris(+), TMA(+) and TEA(+) are virtually impermeant. It is likely thatthe ionic composition of MMEBSS will establish conditions that willpermit Ca⁺⁺ ions to pass through the channel in sufficient quantities touse a calcium influx assay to measure channel activity. Under theseconditions, a calcium influx assay can be used as a high throughputassay using P2X receptors as a drug target.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art, all of which are intended as aspects of theinvention. Accordingly only such limitations as appear in the claimsshould be placed in the invention.

14 1 1509 DNA Homo sapiens 1 atgcgctgct cgccgggagg cgtctggctg gcgctggccgcgtcgctcct gcacgtgtcc 60 ctgcaaggcg agttccagag gaagctttac aaggagctggtcaagaacta caatcccttg 120 gagaggcccg tggccaatga ctcgcaacca ctcaccgtctacttctccct gagcctcctg 180 cagatcatgg acgtggatga gaagaaccaa gttttaaccaccaacatttg gctgcaaatg 240 tcttggacag atcactattt acagtggaat gtgtcagaatatccaggggt gaagactgtt 300 cgtttcccag atggccagat ttggaaacca gacattcttctctataacag tgctgatgag 360 cgctttgacg ccacattcca cactaacgtg ttggtgaattcttctgggca ttgccagtac 420 ctgcctccag gcatattcaa gagttcctgc tacatcgatgtacgctggtt tccctttgat 480 gtgcagcact gcaaactgaa gtttgggtcc tggtcttacggaggctggtc cttggatctg 540 cagatgcagg aggcagatat cagtggctat atccccaatggagaatggga cctagtggga 600 atccccggca agaggagtga aaggttctat gagtgctgcaaagagcccta ccccgatgtc 660 accttcacag tgaccatgcg ccgcaggacg ctctactatggcctcaacct gctgatcccc 720 tgtgtgctca tctccgccct cgccctgctg gtgttcctgcttcctgcaga ttccggggag 780 aagatttccc tggggataac agtcttactc tctcttaccgtcttcatgct gctcgtggct 840 gagatcatgc ccgcaacatc cgattcggta ccattgatagcccagtactt cgccagcacc 900 atgatcatcg tgggcctctc ggtggtggtg acggtgatcgtgctgcagta ccaccaccac 960 gaccccgacg ggggcaagat gcccaagtgg accagagtcatccttctgaa ctggtgcgcg 1020 tggttcctgc gaatgaagag gcccggggag gacaaggtgcgcccggcctg ccagcacaag 1080 cagcggcgct gcagcctggc cagtgtggag atgagcgccgtggcgccgcc gcccgccagc 1140 aacgggaacc tgctgtacat cggcttccgc ggcctggacggcgtgcactg tgtcccgacc 1200 cccgactctg gggtagtgtg tggccgcatg gcctgctcccccacgcacga tgagcacctc 1260 ctgcacggcg ggcaaccccc cgagggggac ccggacttggccaagatcct ggaggaggtc 1320 cgctacattg ccaatcgctt ccgctgccag gacgaaagcgaggcggtctg cagcgagtgg 1380 aagttcgccg cctgtgtggt ggaccgcctg tgcctcatggccttctcggt cttcaccatc 1440 atctgcacca tcggcatcct gatgtcggct cccaacttcgtggaggccgt gtccaaagac 1500 tttgcgtaa 1509 2 502 PRT Homo sapiens 2 MetArg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 7580 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 9095 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro ProGly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe ProPhe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp SerTyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp IleSer Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile ProGly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro TyrPro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Thr Leu TyrTyr Gly Leu Asn Leu Leu Ile Pro 225 230 235 240 Cys Val Leu Ile Ser AlaLeu Ala Leu Leu Val Phe Leu Leu Pro Ala 245 250 255 Asp Ser Gly Glu LysIle Ser Leu Gly Ile Thr Val Leu Leu Ser Leu 260 265 270 Thr Val Phe MetLeu Leu Val Ala Glu Ile Met Pro Ala Thr Ser Asp 275 280 285 Ser Val ProLeu Ile Ala Gln Tyr Phe Ala Ser Thr Met Ile Ile Val 290 295 300 Gly LeuSer Val Val Val Thr Val Ile Val Leu Gln Tyr His His His 305 310 315 320Asp Pro Asp Gly Gly Lys Met Pro Lys Trp Thr Arg Val Ile Leu Leu 325 330335 Asn Trp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro Gly Glu Asp Lys 340345 350 Val Arg Pro Ala Cys Gln His Lys Gln Arg Arg Cys Ser Leu Ala Ser355 360 365 Val Glu Met Ser Ala Val Ala Pro Pro Pro Ala Ser Asn Gly AsnLeu 370 375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Asp Gly Val His Cys ValPro Thr 385 390 395 400 Pro Asp Ser Gly Val Val Cys Gly Arg Met Ala CysSer Pro Thr His 405 410 415 Asp Glu His Leu Leu His Gly Gly Gln Pro ProGlu Gly Asp Pro Asp 420 425 430 Leu Ala Lys Ile Leu Glu Glu Val Arg TyrIle Ala Asn Arg Phe Arg 435 440 445 Cys Gln Asp Glu Ser Glu Ala Val CysSer Glu Trp Lys Phe Ala Ala 450 455 460 Cys Val Val Asp Arg Leu Cys LeuMet Ala Phe Ser Val Phe Thr Ile 465 470 475 480 Ile Cys Thr Ile Gly IleLeu Met Ser Ala Pro Asn Phe Val Glu Ala 485 490 495 Val Ser Lys Asp PheAla 500 3 1464 DNA Mus musculus 3 atgcggctct gcatcccgca ggtgctgttggccttgttcc tttccatgct gacagccccg 60 ggagaaggca gccggaggag ggccacccaggaggatacca cccagcctgc tctactaagg 120 ctgtcagacc acctcctggc taactacaagaagggggtgc ggcctgtgcg ggactggagg 180 aagcctacta ctgtctccat tgatgtcatcatgtatgcca tcctcaacgt ggatgagaag 240 aaccaggttc tgaccaccta catatggtaccggcagtact ggactgatga gtttctgcag 300 tggactcctg aggacttcga caatgtcaccaaattgtcca tccccacaga cagcatctgg 360 gtccctgaca ttctcatcaa tgagtttgtggacgtgggga agtctccgaa cattccttat 420 gtgtacgtgc atcatcgagg tgaagttcagaactacaagc ccttgcaatt ggtgaccgcc 480 tgtagccttg acatctacaa cttcccctttgatgtgcaga actgttctct gactttcacc 540 agctggctgc acaccatcca ggacatcaacattactctgt ggcgatcacc ggaagaagtg 600 aggtctgaca agagcatctt cataaatcagggcgagtggg agctgctgga ggtgttcccc 660 cagttcaagg agttcagcat agatatcagtaacagctatg cagaaatgaa gttctacgtg 720 atcatccgcc ggaggccttt attctatgcagtcagcctct tgctgcccag tatcttcctc 780 atggtcgtgg acattgtggg cttttgcctgcccccggaca gtggtgagag agtctctttc 840 aagatcacac tccttctggg atactcagtcttcctcatca tcgtgtcaga cacactgccg 900 gcaacgatcg gtacccccct cattggtgtctactttgtgg tgtgcatggc tctgctagtg 960 ataagcctcg ctgagaccat cttcattgtgcggctggtgc ataagcagga cctacagcgg 1020 ccagtacctg actggctgag gcacctggtcctagacagaa tagcctggat actctgccta 1080 ggggagcagc ctatggccca tagacccccagccaccttcc aagccaacaa gactgatgac 1140 tgctcaggtt ctgatcttct tccagccatgggaaaccact gcagccatgt tggaggacct 1200 caggacttgg agaagacccc aaggggcagaggtagccctc ttccaccacc aagggaggcc 1260 tcactggctg tgcgtggtct cttgcaagagctatcctcca tccgccactt cctggagaag 1320 cgggatgaga tgcgggaggt ggcaagggactggctgcggg tgggatacgt gctggacagg 1380 ctgctgttcc gcatctacct gctggctgtgctcgcttaca gcatcaccct ggtcactctc 1440 tggtccattt ggcattattc ttga 1464 4457 PRT Mus musculus 4 Glu Asp Thr Thr Gln Pro Ala Leu Leu Arg Leu SerAsp His Leu Leu 1 5 10 15 Ala Asn Tyr Lys Lys Gly Val Arg Pro Val ArgAsp Trp Arg Lys Pro 20 25 30 Thr Thr Val Ser Ile Asp Val Ile Met Tyr AlaIle Leu Asn Val Asp 35 40 45 Glu Lys Asn Gln Val Leu Thr Thr Tyr Ile TrpTyr Arg Gln Tyr Trp 50 55 60 Thr Asp Glu Phe Leu Gln Trp Thr Pro Glu AspPhe Asp Asn Val Thr 65 70 75 80 Lys Leu Ser Ile Pro Thr Asp Ser Ile TrpVal Pro Asp Ile Leu Ile 85 90 95 Asn Glu Phe Val Asp Val Gly Lys Ser ProAsn Ile Pro Tyr Val Tyr 100 105 110 Val His His Arg Gly Glu Val Gln AsnTyr Lys Pro Leu Gln Leu Val 115 120 125 Thr Ala Cys Ser Leu Asp Ile TyrAsn Phe Pro Phe Asp Val Gln Asn 130 135 140 Cys Ser Leu Thr Phe Thr SerTrp Leu His Thr Ile Gln Asp Ile Asn 145 150 155 160 Ile Thr Leu Trp ArgSer Pro Glu Glu Val Arg Ser Asp Lys Ser Ile 165 170 175 Phe Ile Asn GlnGly Glu Trp Glu Leu Leu Glu Val Phe Pro Gln Phe 180 185 190 Lys Glu PheSer Ile Asp Ile Ser Asn Ser Tyr Ala Glu Met Lys Phe 195 200 205 Tyr ValIle Ile Arg Arg Arg Pro Leu Phe Tyr Ala Val Ser Leu Leu 210 215 220 LeuPro Ser Ile Phe Leu Met Val Val Asp Ile Val Gly Phe Cys Leu 225 230 235240 Pro Pro Asp Ser Gly Glu Arg Val Ser Phe Lys Ile Thr Leu Leu Leu 245250 255 Gly Tyr Ser Val Phe Leu Ile Ile Val Ser Asp Thr Leu Pro Ala Thr260 265 270 Ile Gly Thr Pro Leu Ile Gly Val Tyr Phe Val Val Cys Met AlaLeu 275 280 285 Leu Val Ile Ser Leu Ala Glu Thr Ile Phe Ile Val Arg LeuVal His 290 295 300 Lys Gln Asp Leu Gln Arg Pro Val Pro Asp Trp Leu ArgHis Leu Val 305 310 315 320 Leu Asp Arg Ile Ala Trp Ile Leu Cys Leu GlyGlu Gln Pro Met Ala 325 330 335 His Arg Pro Pro Ala Thr Phe Gln Ala AsnLys Thr Asp Asp Cys Ser 340 345 350 Gly Ser Asp Leu Leu Pro Ala Met GlyAsn His Cys Ser His Val Gly 355 360 365 Gly Pro Gln Asp Leu Glu Lys ThrPro Arg Gly Arg Gly Ser Pro Leu 370 375 380 Pro Pro Pro Arg Glu Ala SerLeu Ala Val Arg Gly Leu Leu Gln Glu 385 390 395 400 Leu Ser Ser Ile ArgHis Phe Leu Glu Lys Arg Asp Glu Met Arg Glu 405 410 415 Val Ala Arg AspTrp Leu Arg Val Gly Tyr Val Leu Asp Arg Leu Leu 420 425 430 Phe Arg IleTyr Leu Leu Ala Val Leu Ala Tyr Ser Ile Thr Leu Val 435 440 445 Thr LeuTrp Ser Ile Trp His Tyr Ser 450 455 5 1416 DNA Artificial SequenceDescription of Artificial Sequence human/mouse hybrid sequence 5atgcgctgct cgccgggagg cgtctggctg gcgctggccg cgtcgctcct gcacgtgtcc 60ctgcaaggcg agttccagag gaagctttac aaggagctgg tcaagaacta caatcccttg 120gagaggcccg tggccaatga ctcgcaacca ctcaccgtct acttctccct gagcctcctg 180cagatcatgg acgtggatga gaagaaccaa gttttaacca ccaacatttg gctgcaaatg 240tcttggacag atcactattt acagtggaat gtgtcagaat atccaggggt gaagactgtt 300cgtttcccag atggccagat ttggaaacca gacattcttc tctataacag tgctgatgag 360cgctttgacg ccacattcca cactaacgtg ttggtgaatt cttctgggca ttgccagtac 420ctgcctccag gcatattcaa gagttcctgc tacatcgatg tacgctggtt tccctttgat 480gtgcagcact gcaaactgaa gtttgggtcc tggtcttacg gaggctggtc cttggatctg 540cagatgcagg aggcagatat cagtggctat atccccaatg gagaatggga cctagtggga 600atccccggca agaggagtga aaggttctat gagtgctgca aagagcccta ccccgatgtc 660accttcacag tgaccatgcg ccgcaggacg ttattctatg cagtcagcct cttgctgccc 720agtatcttcc tcatggtcgt ggacattgtg ggcttttgcc tgcccccgga cagtggtgag 780agagtctctt tcaagatcac actccttctg ggatactcag tcttcctcat catcgtgtca 840gacacactgc cggcaacgat cggtaccccc ctcattggtg tctactttgt ggtgtgcatg 900gctctgctag tgataagcct cgctgagacc atcttcattg tgcggctggt gcataagcag 960gacctacagc ggccagtacc tgactggctg aggcacctgg tcctagacag aatagcctgg 1020atactctgcc taggggagca gcctatggcc catagacccc cagccacctt ccaagccaac 1080aagactgatg actgctcagg ttctgatctt cttccagcca tgggaaacca ctgcagccat 1140gttggaggac ctcaggactt ggagaagacc ccaaggggca gaggtagccc tcttccacca 1200ccaagggagg cctcactggc tgtgcgtggt ctcttgcaag agctatcctc catccgccac 1260ttcctggaga agcgggatga gatgcgggag gtggcaaggg actggctgcg ggtgggatac 1320gtgctggaca ggctgctgtt ccgcatctac ctgctggctg tgctcgctta cagcatcacc 1380ctggtcactc tctggtccat ttggcattat tcttga 1416 6 470 PRT ArtificialSequence Description of Artificial Sequence human/mouse hybrid sequence6 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 1015 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 2530 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 4045 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 5560 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 7075 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 8590 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe HisThr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu ProPro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp PhePro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser TrpSer Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala AspIle Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly IlePro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu ProTyr Pro Asp Val Thr Phe Thr Val 210 215 220 Ile Ile Arg Arg Arg Pro PheTyr Ala Val Ser Leu Leu Leu Pro Ser 225 230 235 240 Ile Phe Leu Met ValVal Asp Ile Val Gly Phe Cys Leu Pro Pro Asp 245 250 255 Ser Gly Glu ArgVal Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr Ser 260 265 270 Val Phe LeuIle Ile Val Ser Asp Thr Leu Pro Ala Thr Ile Gly Thr 275 280 285 Pro LeuIle Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu Val Ile 290 295 300 SerLeu Ala Glu Thr Ile Phe Ile Val Arg Leu Val His Lys Gln Asp 305 310 315320 Leu Gln Arg Pro Val Pro Asp Trp Leu Arg His Leu Val Leu Asp Arg 325330 335 Ile Ala Trp Ile Leu Cys Leu Gly Glu Gln Pro Met Ala His Arg Pro340 345 350 Pro Ala Thr Phe Gln Ala Asn Lys Thr Asp Asp Cys Ser Gly SerAsp 355 360 365 Leu Leu Pro Ala Met Gly Asn His Cys Ser His Val Gly GlyPro Gln 370 375 380 Asp Leu Glu Lys Thr Pro Arg Gly Arg Gly Ser Pro LeuPro Pro Pro 385 390 395 400 Arg Glu Ala Ser Leu Ala Val Arg Gly Leu LeuGln Glu Leu Ser Ser 405 410 415 Ile Arg His Phe Leu Glu Lys Arg Asp GluMet Arg Glu Val Ala Arg 420 425 430 Asp Trp Leu Arg Val Gly Tyr Val LeuAsp Arg Leu Leu Phe Arg Ile 435 440 445 Tyr Leu Leu Ala Val Leu Ala TyrSer Ile Thr Leu Val Thr Leu Trp 450 455 460 Ser Ile Trp His Tyr Ser 465470 7 44 DNA Artificial Sequence Description of Artificial SequenceGG443PCR Primer 7 ggctctagac caccatgcgc tgttcaccgg gaggcgtctg gctg 44 8 37DNA Artificial Sequence Description of Artificial SequenceGG444 PCRPrimer 8 gggtgatcac tgtgaaggtg acatcagggt agggctc 37 9 1509 DNA Homosapiens 9 atgcgctgct cgccgggagg cgtctggctg gcgctggccg cgtcgctcctgcacgtgtcc 60 ctgcaaggcg agttccagag gaagctttac aaggagctgg tcaagaactacaatcccttg 120 gagaggcccg tggccaatga ctcgcaacca ctcaccgtct acttctccctgagcctcctg 180 cagatcatgg acgtggatga gaagaaccaa gttttaacca ccaacatttggctgcaaatg 240 tcttggacag atcactattt acagtggaat gtgtcagaat atccaggggtgaagactgtt 300 cgtttcccag atggccagat ttggaaacca gacattcttc tctataacagtgctgatgag 360 cgctttgacg ccacattcca cactaacgtg ttggtgaatt cttctgggcattgccagtac 420 ctgcctccag gcatattcaa gagttcctgc tacatcgatg tacgctggtttccctttgat 480 gtgcagcact gcaaactgaa gtttgggtcc tggtcttacg gaggctggtccttggatctg 540 cagatgcagg aggcagatat cagtggctat atccccaatg gagaatgggacctagtggga 600 atccccggca agaggagtga aaggttctat gagtgctgca aagagccctaccccgatgtc 660 accttcacag tgaccatgcg ccgcaggccg ctctactatg gcctcaacctgctgatcccc 720 tgtgtgctca tctccgccct cgccctgctg gtgttcctgc ttcctgcagattccggggag 780 aagatttccc tggggataac agtcttactc tctcttaccg tcttcatgctgctcgtggct 840 gagatcatgc ccgcaacatc cgattcggta ccattgatag cccagtacttcgccagcacc 900 atgatcatcg tgggcctctc ggtggtggtg acggtgatcg tgctgcagtaccaccaccac 960 gaccccgacg ggggcaagat gcccaagtgg accagagtca tccttctgaactggtgcgcg 1020 tggttcctgc gaatgaagag gcccggggag gacaaggtgc gcccggcctgccagcacaag 1080 cagcggcgct gcagcctggc cagtgtggag atgagcgccg tggcgccgccgcccgccagc 1140 aacgggaacc tgctgtacat cggcttccgc ggcctggacg gcgtgcactgtgtcccgacc 1200 cccgactctg gggtagtgtg tggccgcatg gcctgctccc ccacgcacgatgagcacctc 1260 ctgcacggcg ggcaaccccc cgagggggac ccggacttgg ccaagatcctggaggaggtc 1320 cgctacattg ccaatcgctt ccgctgccag gacgaaagcg aggcggtctgcagcgagtgg 1380 aagttcgccg cctgtgtggt ggaccgcctg tgcctcatgg ccttctcggtcttcaccatc 1440 atctgcacca tcggcatcct gatgtcggct cccaacttcg tggaggccgtgtccaaagac 1500 tttgcgtaa 1509 10 502 PRT Homo sapiens 10 Met Arg CysSer Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu HisVal Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu ValLys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln ProLeu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val AspGlu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 SerTrp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 ValLys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr GlyGly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser GlyTyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly LysArg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro AspVal Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Pro Leu Tyr Tyr GlyLeu Asn Leu Leu Ile Pro 225 230 235 240 Cys Val Leu Ile Ser Ala Leu AlaLeu Leu Val Phe Leu Leu Pro Ala 245 250 255 Asp Ser Gly Glu Lys Ile SerLeu Gly Ile Thr Val Leu Leu Ser Leu 260 265 270 Thr Val Phe Met Leu LeuVal Ala Glu Ile Met Pro Ala Thr Ser Asp 275 280 285 Ser Val Pro Leu IleAla Gln Tyr Phe Ala Ser Thr Met Ile Ile Val 290 295 300 Gly Leu Ser ValVal Val Thr Val Ile Val Leu Gln Tyr His His His 305 310 315 320 Asp ProAsp Gly Gly Lys Met Pro Lys Trp Thr Arg Val Ile Leu Leu 325 330 335 AsnTrp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro Gly Glu Asp Lys 340 345 350Val Arg Pro Ala Cys Gln His Lys Gln Arg Arg Cys Ser Leu Ala Ser 355 360365 Val Glu Met Ser Ala Val Ala Pro Pro Pro Ala Ser Asn Gly Asn Leu 370375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Asp Gly Val His Cys Val Pro Thr385 390 395 400 Pro Asp Ser Gly Val Val Cys Gly Arg Met Ala Cys Ser ProThr His 405 410 415 Asp Glu His Leu Leu His Gly Gly Gln Pro Pro Glu GlyAsp Pro Asp 420 425 430 Leu Ala Lys Ile Leu Glu Glu Val Arg Tyr Ile AlaAsn Arg Phe Arg 435 440 445 Cys Gln Asp Glu Ser Glu Ala Val Cys Ser GluTrp Lys Phe Ala Ala 450 455 460 Cys Val Val Asp Arg Leu Cys Leu Met AlaPhe Ser Val Phe Thr Ile 465 470 475 480 Ile Cys Thr Ile Gly Ile Leu MetSer Ala Pro Asn Phe Val Glu Ala 485 490 495 Val Ser Lys Asp Phe Ala 50011 1509 DNA Homo sapiens 11 atgcgctgct cgccgggagg cgtctggctg gcgctggccgcgtcgctcct gcacgtgtcc 60 ctgcaaggcg agttccagag gaagctttac aaggagctggtcaagaacta caatcccttg 120 gagaggcccg tggccaatga ctcgcaacca ctcaccgtctacttctccct gagcctcctg 180 cagatcatgg acgtggatga gaagaaccaa gttttaaccaccaacatttg gctgcaaatg 240 tcttggacag atcactattt acagtggaat gtgtcagaatatccaggggt gaagactgtt 300 cgtttcccag atggccagat ttggaaacca gacattcttctctataacag tgctgatgag 360 cgctttgacg ccacattcca cactaacgtg ttggtgaattcttctgggca ttgccagtac 420 ctgcctccag gcatattcaa gagttcctgc tacatcgatgtacgctggtt tccctttgat 480 gtgcagcact gcaaactgaa gtttgggtcc tggtcttacggaggctggtc cttggatctg 540 cagatgcagg aggcagatat cagtggctat atccccaatggagaatggga cctagtggga 600 atccccggca agaggagtga aaggttctat gagtgctgcaaagagcccta ccccgatgtc 660 accttcacag tgaccatgcg ccgcaggacg ctctactatggcctcaacct gctgatcccc 720 agtgtgctca tctccgccct cgccctgctg gtgttcctgcttcctgcaga ttccggggag 780 aagatttccc tggggataac agtcttactc tctcttaccgtcttcatgct gctcgtggct 840 gagatcatgc ccgcaacatc cgattcggta ccattgatagcccagtactt cgccagcacc 900 atgatcatcg tgggcctctc ggtggtggtg acggtgatcgtgctgcagta ccaccaccac 960 gaccccgacg ggggcaagat gcccaagtgg accagagtcatccttctgaa ctggtgcgcg 1020 tggttcctgc gaatgaagag gcccggggag gacaaggtgcgcccggcctg ccagcacaag 1080 cagcggcgct gcagcctggc cagtgtggag atgagcgccgtggcgccgcc gcccgccagc 1140 aacgggaacc tgctgtacat cggcttccgc ggcctggacggcgtgcactg tgtcccgacc 1200 cccgactctg gggtagtgtg tggccgcatg gcctgctcccccacgcacga tgagcacctc 1260 ctgcacggcg ggcaaccccc cgagggggac ccggacttggccaagatcct ggaggaggtc 1320 cgctacattg ccaatcgctt ccgctgccag gacgaaagcgaggcggtctg cagcgagtgg 1380 aagttcgccg cctgtgtggt ggaccgcctg tgcctcatggccttctcggt cttcaccatc 1440 atctgcacca tcggcatcct gatgtcggct cccaacttcgtggaggccgt gtccaaagac 1500 tttgcgtaa 1509 12 502 PRT Homo sapiens 12 MetArg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 7580 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 9095 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro ProGly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe ProPhe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp SerTyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp IleSer Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile ProGly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro TyrPro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Thr Leu TyrTyr Gly Leu Asn Leu Leu Ile Pro 225 230 235 240 Ser Val Leu Ile Ser AlaLeu Ala Leu Leu Val Phe Leu Leu Pro Ala 245 250 255 Asp Ser Gly Glu LysIle Ser Leu Gly Ile Thr Val Leu Leu Ser Leu 260 265 270 Thr Val Phe MetLeu Leu Val Ala Glu Ile Met Pro Ala Thr Ser Asp 275 280 285 Ser Val ProLeu Ile Ala Gln Tyr Phe Ala Ser Thr Met Ile Ile Val 290 295 300 Gly LeuSer Val Val Val Thr Val Ile Val Leu Gln Tyr His His His 305 310 315 320Asp Pro Asp Gly Gly Lys Met Pro Lys Trp Thr Arg Val Ile Leu Leu 325 330335 Asn Trp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro Gly Glu Asp Lys 340345 350 Val Arg Pro Ala Cys Gln His Lys Gln Arg Arg Cys Ser Leu Ala Ser355 360 365 Val Glu Met Ser Ala Val Ala Pro Pro Pro Ala Ser Asn Gly AsnLeu 370 375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Asp Gly Val His Cys ValPro Thr 385 390 395 400 Pro Asp Ser Gly Val Val Cys Gly Arg Met Ala CysSer Pro Thr His 405 410 415 Asp Glu His Leu Leu His Gly Gly Gln Pro ProGlu Gly Asp Pro Asp 420 425 430 Leu Ala Lys Ile Leu Glu Glu Val Arg TyrIle Ala Asn Arg Phe Arg 435 440 445 Cys Gln Asp Glu Ser Glu Ala Val CysSer Glu Trp Lys Phe Ala Ala 450 455 460 Cys Val Val Asp Arg Leu Cys LeuMet Ala Phe Ser Val Phe Thr Ile 465 470 475 480 Ile Cys Thr Ile Gly IleLeu Met Ser Ala Pro Asn Phe Val Glu Ala 485 490 495 Val Ser Lys Asp PheAla 500 13 1509 DNA Homo sapiens 13 atgcgctgct cgccgggagg cgtctggctggcgctggccg cgtcgctcct gcacgtgtcc 60 ctgcaaggcg agttccagag gaagctttacaaggagctgg tcaagaacta caatcccttg 120 gagaggcccg tggccaatga ctcgcaaccactcaccgtct acttctccct gagcctcctg 180 cagatcatgg acgtggatga gaagaaccaagttttaacca ccaacatttg gctgcaaatg 240 tcttggacag atcactattt acagtggaatgtgtcagaat atccaggggt gaagactgtt 300 cgtttcccag atggccagat ttggaaaccagacattcttc tctataacag tgctgatgag 360 cgctttgacg ccacattcca cactaacgtgttggtgaatt cttctgggca ttgccagtac 420 ctgcctccag gcatattcaa gagttcctgctacatcgatg tacgctggtt tccctttgat 480 gtgcagcact gcaaactgaa gtttgggtcctggtcttacg gaggctggtc cttggatctg 540 cagatgcagg aggcagatat cagtggctatatccccaatg gagaatggga cctagtggga 600 atccccggca agaggagtga aaggttctatgagtgctgca aagagcccta ccccgatgtc 660 accttcacag tgaccatgcg ccgcaggccgctctactatg gcctcaacct gctgatcccc 720 agtgtgctca tctccgccct cgccctgctggtgttcctgc ttcctgcaga ttccggggag 780 aagatttccc tggggataac agtcttactctctcttaccg tcttcatgct gctcgtggct 840 gagatcatgc ccgcaacatc cgattcggtaccattgatag cccagtactt cgccagcacc 900 atgatcatcg tgggcctctc ggtggtggtgacggtgatcg tgctgcagta ccaccaccac 960 gaccccgacg ggggcaagat gcccaagtggaccagagtca tccttctgaa ctggtgcgcg 1020 tggttcctgc gaatgaagag gcccggggaggacaaggtgc gcccggcctg ccagcacaag 1080 cagcggcgct gcagcctggc cagtgtggagatgagcgccg tggcgccgcc gcccgccagc 1140 aacgggaacc tgctgtacat cggcttccgcggcctggacg gcgtgcactg tgtcccgacc 1200 cccgactctg gggtagtgtg tggccgcatggcctgctccc ccacgcacga tgagcacctc 1260 ctgcacggcg ggcaaccccc cgagggggacccggacttgg ccaagatcct ggaggaggtc 1320 cgctacattg ccaatcgctt ccgctgccaggacgaaagcg aggcggtctg cagcgagtgg 1380 aagttcgccg cctgtgtggt ggaccgcctgtgcctcatgg ccttctcggt cttcaccatc 1440 atctgcacca tcggcatcct gatgtcggctcccaacttcg tggaggccgt gtccaaagac 1500 tttgcgtaa 1509 14 502 PRT Homosapiens 14 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala SerLeu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu TyrLys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala AsnAsp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln IleMet Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp LeuGln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser GluTyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp LysPro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp AlaThr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys GlnTyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp ValArg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys PheGly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met GlnGlu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp LeuVal Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys CysLys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg ArgArg Pro Leu Tyr Tyr Gly Leu Asn Leu Leu Ile Pro 225 230 235 240 Ser ValLeu Ile Ser Ala Leu Ala Leu Leu Val Phe Leu Leu Pro Ala 245 250 255 AspSer Gly Glu Lys Ile Ser Leu Gly Ile Thr Val Leu Leu Ser Leu 260 265 270Thr Val Phe Met Leu Leu Val Ala Glu Ile Met Pro Ala Thr Ser Asp 275 280285 Ser Val Pro Leu Ile Ala Gln Tyr Phe Ala Ser Thr Met Ile Ile Val 290295 300 Gly Leu Ser Val Val Val Thr Val Ile Val Leu Gln Tyr His His His305 310 315 320 Asp Pro Asp Gly Gly Lys Met Pro Lys Trp Thr Arg Val IleLeu Leu 325 330 335 Asn Trp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro GlyGlu Asp Lys 340 345 350 Val Arg Pro Ala Cys Gln His Lys Gln Arg Arg CysSer Leu Ala Ser 355 360 365 Val Glu Met Ser Ala Val Ala Pro Pro Pro AlaSer Asn Gly Asn Leu 370 375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Asp GlyVal His Cys Val Pro Thr 385 390 395 400 Pro Asp Ser Gly Val Val Cys GlyArg Met Ala Cys Ser Pro Thr His 405 410 415 Asp Glu His Leu Leu His GlyGly Gln Pro Pro Glu Gly Asp Pro Asp 420 425 430 Leu Ala Lys Ile Leu GluGlu Val Arg Tyr Ile Ala Asn Arg Phe Arg 435 440 445 Cys Gln Asp Glu SerGlu Ala Val Cys Ser Glu Trp Lys Phe Ala Ala 450 455 460 Cys Val Val AspArg Leu Cys Leu Met Ala Phe Ser Val Phe Thr Ile 465 470 475 480 Ile CysThr Ile Gly Ile Leu Met Ser Ala Pro Asn Phe Val Glu Ala 485 490 495 ValSer Lys Asp Phe Ala 500

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence set forth in SEQ ID NO:14.
 2. An isolated polypeptidecomprising residues 23 through 502 of SEQ ID NO:14.